Tze-Wen Chung

The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin-polysaccharide cardiac patches in vitro.

Polysaccharides and proteins profoundly impact the development and growth of tissues in the natural extra-cellular matrix (ECM). To mimic a natural ECM, polysaccharides were incorporated to/or co-sprayed with silk fibroin (SF) to produce SF/chitosan (CS) or SF/CS-hyaluronic acid (SF/CS-HA) microparticles that were further processed by mechanical pressing and genipin cross-linking to produce hybrid cardiac patches. The ATR-FTIR spectra confirm the co-existence of CS or CS-HA and SF in microparticles and patches. For evaluating the cellular responses of rMSCs to the SF/CS and SF/CS-HA cardiac patches, the growth of rMSCs and cardiomyogenic differentiation of 5-aza inducing rMSCs cultured on patches was examined. First, the isolated rMSCs were identified with various positive and negative surface markers such as CD 44 and CD 31 by a flow cytometric technique, respectively. For examining the growth of rMSCs on the patches, MTT viability assay was performed, and the results demonstrated that the growth of rMSCs on SF and SF-hybrid patches significantly exceeded (P<0.001) that on culture wells after seven days of cultivation. Additionally, the relative growth rates of rMSCs on SF/CS and SF/CS-HA hybrid patches were significantly better (P<0.01) than that on SF patches that were also observed by using vimentin stain to the cells. For instance, the relative cell growth rates (%) in cell culture wells, SF, SF/CS and SF/CS-HA patches were 100%, 282.9+/-6.5%, 337.0+/-8.0% and 332.6+/-6.6% (n=6, for all), respectively. For investigating the effects of the hybrid patches on cardiomyogenic differentiation of 5-aza inducing rMSCs, the expressions of specific cardiac genes of cells such as Gata4 and Nkx2.5 were examined by real-time quantitative polymerase chain reaction (real-time PCR) analysis. The results of cardiomyogenic differentiation of induced rMSCs on SF/CS and SF/CS-HA hybrid patches significantly improved the expressions of cardiac genes of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) than those on SF patches and culture wells. Interestingly, the results of cardiac gene expressions of the cells on the SF/CS-HA hybrid patches were the most pronounced in promoting cardiomyogenic differentiations in this investigation. Furthermore, immunofluorescence staining of cardiac proteins such as cardiotin and connexin 43 for induced rMSCs cultured on SF/CS and SF/CS-HA hybrid patches were much pronounced compared with SF patches, indicating the improvements of cardiomyogenic differentiation on the hybrid patches. The results of this study demonstrate that the SF/CS and SF/CS-HA hybrid patches may be promising biomaterials for regenerating infarcted cardiac tissues.

Activity-dependent neuroprotector homeobox protein: A candidate protein identified in serum as diagnostic biomarker for Alzheimer's disease

Alzheimers disease (AD) is the most common cause of dementia of late life. To enhance our understanding of AD proteome, the serum proteins were analyzed using two-dimensional gel electrophoresis (2DE) combined with nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) followed by peptide fragmentation patterning. In this study, six protein spots with differential expression were identified. Five up-regulated proteins were identified as actin, apolipoprotein A-IV (Apo A-IV), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-1-antitrypsin (AAT), and antithrombin-III (AT-III); one protein, activity-dependent neuroprotector homeobox protein (ADNP) was down-regulated in AD patients. These proteins with differential expression in the serum may serve as potential indicators of AD. Our results suggested that ADNP may play an important role in slowing the progression of clinical symptoms of AD.

Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model.

Bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid (BMSC/SH) patches were implanted into myocardial infarction (MI) rat hearts to investigate the efficacies of them on enhancing left ventricular (LV) remodeling and cardiac repair. 45 rats were divided into four groups: Sham, MI (MI hearts, induced by a cryo-injury technique), SH and BMSC/SH (MI hearts with implantations of SH and BMSC/SH patches, respectively). After eight weeks of post-implantation, the patches for the SH and BMSC/SH groups were intact and well adhered on the MI zones with no and minor immunological responses, respectively, examined by a CD68 marker, while severe inflammation on the zones was observed for the MI group. The SH group showed the efficacy of cardiac repair on MI zones. Moreover, BMSC/SH group significantly improved the wall thickness of LV, assessed by echocardiography, and had high viability of delivery BMSC, largely reduced apoptosis, significantly promoted neo-vascularization and stimulated the secretions of various paracrine factors such as VEGF, examined by real-time PCR, in MI zones compared with those of the SH and MI groups. In conclusion, the therapeutic efficacies of using BMSC/SH patches for repairing MI hearts were demonstrated by showing the advantages of both bioactive SH patches and BMSC-based therapy.

The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin – Hyaluronic acid cardiac patches

Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC (rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5-aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9+/-7.1 (%) or 48.4+/-6.0 (%) (n=3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8x10(5)/cell, in fluorescent intensity) while cells with a CD44-blockage treatment markedly diminished the expressions (e.g., 1.1x10(4)/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore, the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44-blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches.

Promoting regeneration of peripheral nerves in-vivo using new PCL-NGF/Tirofiban nerve conduits

Poly(ε-caprolactone) (PCL) scaffolds were modified by grafting nerve growth factor (NGF) and Tirofiban (TF), a clinical anti-thrombosis drug, as a new biomaterial for producing nerve conduits to promote the regeneration of sciatic nerves. The successful grafting of NGF and TF onto PCL scaffolds was confirmed by FTIR and ESCA spectra. In-vitro growths of the PC12 cells in PCL-NGF and PCL-NGF/TF scaffolds, determined by MTS, were significantly higher (P < 0.05, n = 4) than those in the PCL scaffolds following three days of cultivation. Interestingly, this study evaluation of the PCL, PCL-NGF, and PCL-NGF/TF nerve conduits in a 12 mm long gap of the rat sciatic nerve defect model that the gastrocnemius muscle mass of the tested rats in the PCL-NGF/TF groups significantly exceeded those in the PCL-NGF and PCL group. In the rats that had been implanted with PCL-NGF/TF conduits, the generated nerves passed through those conduits, expressing beta-III tubulin (TB), growth association protein-43 (GAP-43) and myelin basic protein (MBP) along their longitudinal axis, and the proximal and distal nerve ends of the rats were successfully connected. Those that had been implanted with PCL and PCL-NGF conduits did not exhibit these effects, as revealed by an immunochemical study of the expressions of the proteins in the conduits. Moreover, counting within the dorsal horn of the spinal cord (C(5)) demonstrated that the numbers of CTB-HRP-labeled neurons in the rats that had been implanted with PCL-NGF/TF conduits were significantly higher than those in the other groups. In this study, in-vivo examinations of the use of newly designed PCL-NGF/TF conduits to promote the generation of nerves in a defective rat model significantly increased the gastrocnemius muscle mass, and led to the successful regeneration of nerves that bridged a 12 mm long defected gap of nerves in rats. However, more rats must be tested to confirm the efficacy the newly designed nerve conduits.

Effects of the characteristics of chitosan on controlling drug release of chitosan coated PLLA microspheres.

Chitosan has been shown to be a biomaterial with good biocompatibility, and is highly biodegradable. This study investigated the effect of post-coating PLLA microspheres with different chitosans on the initial burst and controlling the drug release of the microspheres. Without chitosan, 19.2% of encapsulated lidocaine would release from PLLA microspheres within the first hour (R1), and the time of 50% release (T50) was 25 h. After the microspheres were coated with chitosan of viscosity (eta) 384 +/- 10cp, R1 and T50 could be reduced and prolonged to 14.6% and 90 h, respectively, for all tested molecular weights (Mw) of chitosan. In the case of the same Mw of chitosan being applied, the efficacy of reducing the initial burst of drug release was higher for a lower degree of deacetylation (D.D.). With chitosan in acetic acid solution, coating the microspheres with high Mw and high viscosity could most effectively reduce the initial burst and control drug release of PLLA microspheres. For example, the microspheres coated with chitosan solution of Mw 800 kDa and eta of 1479 cp, R1 and T50 could be reduced and prolonged to 7.4% and 245 h, respectively. The study indicated that manipulating the viscosity of the chitosan solution was the most important factor in contributing to controlling the drug release of chitosan post-coated PLLA microspheres.

Effects of alginate coated on PLGA microspheres for delivery tetracycline hydrochloride to periodontal pockets.

The effects of alginate coated on tetracycline (Tc) loaded poly (D, L-lactic-co-glycolic acid) (PLGA) microspheres fabricated by double emulsion solvent evaporation technique for local delivery to periodontal pocket were investigated. Alginate coated PLGA microspheres showed smoother surface but enlarged their particle sizes compared with those of uncoated ones. In addition, alginate coated microspheres enhanced Tc encapsulation efficiency (E.E.) from 11.5 ± 0.5% of uncoated ones to 17.9 ± 0.5%. Moreover, all of the coated PLGA microspheres even fabricated at different conditions could prolong Tc release from 9–12 days with 50% or higher in cumulative release of Tc compared with those of uncoated ones. The swelling ratios of PLGA microspheres for alginate coated or uncoated ones, one of the possible mechanisms for enhancing Tc release for the coated ones, were measured. The results showed that 20% or higher in swelling ratio for the coated microspheres at the earlier stage of hydration (e.g. ≤ 24 h) could be an important factor to result in high Tc release compared to the uncoated ones. In conclusion, alginate coated Tc loaded PLGA microspheres could enhance Tc delivery to periodontal pocket by enhancing drug encapsulated efficiency, released quantities and sustained release period compared with uncoated ones.

Cardiac repair using chitosan-hyaluronan/silk fibroin patches in a rat heart model with myocardial infarction.

The cardiac repair of myocardial infarction (MI) hearts of rats using chitosan-hyaluronan/silk fibroin (chitosan-HYA/SF) cardiac patches was examined after eight weeks of implantation. Rats with implantations of chitosan-HYA/SF patches (CHS group) significantly (P<0.05) reduced the dilation of the inner diameter of left ventricle (LV) (4.27 ± 0.29 mm), increased wall thickness of LV (1.5 ± 0.13 mm) and improved the fractional shortening of LV of hearts (LVFS) (42.8 ± 2.4%) compared with those values of LVs of rats without implants (MI group) (e.g., 5.92 ± 0.39 mm, 1.2 ± 0.06 mm and 31.5±1.4%, respectively). Moreover, blood vessel-like structures in MI regions of LVs in the CHS group were widely distributed while none was found in the MI group. The CHS group significantly improved the secretion of paracrine factors, such as VEGF in the MI regions of LVs (P<0.05, n=4), relative to that in the MI group. In conclusion, chitosan-HYA/SF cardiac patches are promising biomaterials for the cardiac repair of MI rat hearts.

The t-PA-encapsulated PLGA nanoparticles shelled with CS or CS-GRGD alter both permeation through and dissolving patterns of blood clots compared with t-PA solution: An in vitro thrombolysis study

Accelerated thrombolysis by pressure-driven permeation has been demonstrated in in vitro and in vivo animal models by using plasminogen activators (PAs) encapsulated liposomes or PEG microparticles. Recent reports have also described acceleration of thrombolysis using tissue type PA (t-PA) encapsulated in PLGA nanoparticles (NPs) coated with chitosan (CS) or CS-GRGD by interactions between the NPs and blood clots. However, the permeation through and dissolving patterns in thrombolysis with the aforementioned microparticles or NPs, which may be clinically relevant to the recovery status of the posttreatments, have not been reported. Therefore, this work studied such phenomena in thrombolysis with t-PA encapsulated in NPs. The t-PA solution and the NPs exhibited distinctly different permeation patterns of dissolved clots. Plasma permeates through clots showed a stream flow or burst flow phenomena when lyzed with NPs shelled with CS or CS-GRGD, respectively, whereas a diffusion pattern was observed in those lyzed with t-PA solution. At the outlet position of clots, the clots dissolved with PLGA/CS and PLGA/CS-GRGD NPs revealed extremely rough surfaces to a depth of 100 mum, indicating that a cross-permeation direction of clot lysis occurred, while those dissolved with t-PA solution showed slightly rough surfaces to a depth of 12 mum. Permeation through and clot dissolution patterns of thrombolysis with t-PA encapsulated in NPs shelled with CS or CS-GRGD distinctly differed from those dissolved with t-PA solutions in this in vitro thrombolysis model, These findings may be relevant to posttreatment of patients with conventional PA thrombolysis.

Characterization of ADAM28 as a biomarker of bladder transitional cell carcinomas by urinary proteome analysis

Human urine contains a large number of proteins and peptides (the urinary proteome). Global analysis of the human urinary proteome is important for understanding urinary tract diseases. Bladder cancer is the most common urological cancer with higher incidence rates in endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to use the proteomic approach to establish urinary protein biomarkers of bladder cancer. ADAM28, identified by proteomic approaches and confirmed by Western blotting, showed significant differences compared with normal individuals, so it may be a biomarker of bladder cancer.